拟南芥MTOPVIB与PRD1互作参与减数分裂重组DNA双链断裂的形成

发布时间：2018-05-05 17:59

本文选题：拟南芥 + 减数分裂　；参考：《中国农业大学》2017年博士论文

【摘要】：在显花植物(phanerogams)的有性生殖过程中,雌蕊胚珠和雄蕊花药中的性母细胞(2n)经减数分裂分别形成大孢子(n)和小孢子(n)后,进一步发育分别形成雌配子体(胚囊)和雄配子体(花粉)。减数分裂是雌、雄配子体形成的关键步骤,包括DNA复制、姐妹染色单体黏连、同源染色体配对、联会、重组和分离等一系列重要的生物学事件,任何环节出现异常,都会影响雌雄配子体的形成,导致植物不育。其中,同源染色体重组是遗传信息交换的重要方式,而DNA双链断裂(DNA double-strand break,DSB)的形成是同源重组起始过程,目前已发现几个蛋白参与调控拟南芥DSB的形成,其中AtSPO11-1和AtSP011-2是SPO11的同源蛋白;AtPRD1是与小鼠中MEI1同源但功能未知的蛋白;AtPRD2是小鼠和酵母中Mei4的同源蛋白;AtPRD3和AtDFO是植物所特有但功能未知的DSB形成蛋白。虽然已发现这几个蛋白参与DSB形成过程,但对DSB形成过程的遗传调控机制还缺乏深入了解。因此,研究植物雌、雄配子体的形成以及同源染色体重组的分子遗传机制不仅可以了解植物生殖发育的分子遗传机制,也可为农作物遗传育种提供遗传学方面的理论参考。本研究分离鉴定到了影响拟南芥雌雄配子体发育的突变体mt187,该突变体是一个从基因陷阱(gene-trap)和增强子陷阱(enhancer-trap)Ds插入突变体库中筛选到的。与野生型相比,它的角果短小,育性降低。遗传分析显示,mt187突变体中的Ds插入位点与表型不连锁。图位克隆技术分析显示,突变位点位于At1g60460基因的第八个外显子的最后一个碱基(G1890),该位点G1890A突变造成相关内含子的剪切异常,从而影响该基因的正常表达,并严重影响雌雄配子体的形成,导致植物的育性显著降低。另外,从拟南芥生物资源中心(ABRC)获得的T-DNA插入等位突变体也具有相似的表型。这些研究结果表明,At1g60460基因在雌雄配子体的形成中起重要作用。At1g60460基因编码一个最近被命名为MTOPVIB的蛋白。鉴于已有两个相关的等位突变体,因此把mt187改名为mtopVIB-3。上述T-DNA插入等位突变体与发表在Science中的mtopVIB-2是同一个突变体。相对于mtopVIB-3,T-DNA插入突变体mtopVIB-2具有更严重的表型,其结实率仅有2%。初步观察发现,不仅mtopVIB突变体花药中的四分体发育异常、花粉败育,而且它的胚珠中的雌配子体发育也异常,停滞在FG1期大孢子母细胞时期,暗示可能是雌雄配子体发育过程中的减数分裂过程出现异常。进一步利用染色体展片方法观察发现,与野生型的雄和雌性母细胞减数分裂相比,在粗线期,mtopVIB突变体的染色体没有呈现正常的粗丝状;在终变期,突变体出现单价染色体,而不是正常的五对二价体。同时通过FISH实验和免疫荧光实验证明,突变体的染色体配对、联会出现异常。通过计算突变体的重组率,发现突变体的重组率较野生型显著地下降,表明mtopVIB突变体的同源重组也异常。将表型严重的mtopVIB-2突变体与DSB修复缺陷突变体Atcom1-1、Atrad50-1、Atrad51和Atmre11-1构建相应的双突变体,这些双突变体的表型与mtopVIB-2突变体的表型相似,均在减数分裂过程中出现单价体,表明mtopVIB-2确实影响DSB的形成,说明MTOPVIB参与减数分裂DSB的形成过程。Real-time PCR分析表明,MTOPVIB呈组成型表达,在花序中表达量较高。MTOPVIB蛋白属于一类保守性较高的蛋白,其结构与Topo VI型复合体的B亚基相似,其N端含有一个可以结合并水解ATP的Bergerat结构域,C端含有一个可以诱导构象变化的Transducer结构域。Y2H和BiFC实验结果表明,MTOPVIB蛋白可以分别与AtSPO11-1和AtSPO11-2蛋白互作,而且Y3H和BiFC实验显示,MTOPVIB蛋白可以同时与它们互作,暗示三者间可能形成一个Topo VI类似复合体。Y2H和BiFC实验还表明,AtPRD1可以分别与MTOPVIB和AtSP011-2互作,说明AtPRD1可以与Topo VI类似复合体的各成员互作。利用同样的方法还发现AtPRD1蛋白还可以分别与AtPRD3和AtDFO蛋白互作。此外,Y3H和BiFC实验显示AtPRD1蛋白可以介导与AtPRD3和AtDFO蛋白的互作。这表明AtPRD1很可能是DSB形成过程中的一个重要蛋白。综上所述,在拟南芥中,MTOPVIB蛋白与AtPRD1蛋白互作,并与AtSPO11-1、AtSP011-2、AtPRD3和AtDFO等蛋白共同参与拟南芥雌雄配子体减数分裂过程的DSB形成过程。这些结果为了解拟南芥减数分裂DSB形成的机制提供新的实验证据。
[Abstract]:In the sexual reproduction of phanerogams, the female gametophyte (n) and microspore (n) in the anther of pistil ovule and stamen anther are formed to form a female gametophyte (embryo sac) and male gametophyte (pollen) respectively. Meiosis is a key step in the formation of female and male gametophytes, including DNA replication. A series of important biological events, such as sister chromatid adhesion, homologous chromosome pairing, association, recombination and separation, can affect the formation of male and male gametophytes and cause plant infertility. Among them, homologous chromosome recombination is an important way of genetic information exchange, and DNA double strand breaks (DNA double-strand break, DSB) ) formation is the initiation process of homologous recombination. Several proteins have been found to regulate the formation of Arabidopsis DSB, in which AtSPO11-1 and AtSP011-2 are SPO11 homologous proteins; AtPRD1 is the homologous protein of MEI1 in mice but unknown in function; AtPRD2 is the same source protein of Mei4 in mice and yeast; AtPRD3 and AtDFO are endemic but not functional of plants. The DSB formation protein is known. Although these proteins have been found to be involved in the formation of DSB, the genetic regulation mechanism for the formation of DSB is still lack of understanding. Therefore, the molecular genetic mechanism of the study on the formation of female and male gametophytes and the recombination of homologous chromosomes can not only be used to understand the molecular genetic mechanism of plant reproductive development, but also can be used for the study of the molecular genetic mechanism of plant reproductive development. Genetic breeding for crop genetics is a theoretical reference. This study identified a mutant mt187 that affects the development of male and female gametophyte development in Arabidopsis. This mutant is screened from the gene trap (gene-trap) and enhancer trap (enhancer-trap) Ds insertion mutant library. Compared with the wild type, the mutant is short and fertile. The genetic analysis showed that the Ds insertion site in the mt187 mutant was not linked to the phenotype. The genetic analysis showed that the mutation site was located at the last base of the eighth exons of the At1g60460 gene (G1890), and the G1890A mutation caused the shear anomaly of the related introns, which affected the normal expression of the gene and was serious. In addition, the T-DNA insertion alleles obtained from the Arabidopsis Biological Resource Center (ABRC) also have similar phenotypes. These results suggest that the At1g60460 gene plays a role in the formation of the female and male gametophyte to act as the.At1g60460 gene encoding one recently named as MTOPVIB protein, in view of two related alleles, mt187 was renamed mtopVIB-3. as the same T-DNA insertion mutant and mtopVIB-2 published in Science. Relative to mtopVIB-3, T-DNA insertion mutant mtopVIB-2 has a more serious form, and its seed setting rate is only observed by 2%.. Only four of the mtopVIB mutant anthers have abnormal development, pollen abortion, and the development of the female gametophyte in the ovule is also abnormal, stagnating in the period of the FG1 stage of the megaspore mother cell, suggesting that the meiotic division during the development of the male and the male gametophyte may be abnormal. Compared with the male and female meiosis meiosis, the chromosomes of the mtopVIB mutant did not present normal coarse filamentous chromosomes at the roughing stage. At the final stage, the mutants appeared monovalent chromosomes, but not the normal five to two valence bodies. At the same time, the chromosomal pairing of the mutants appeared to be abnormal through the FISH experiment and immunofluorescence experiments. The recombination rate of the mutant showed that the recombinant rate of the mutant was significantly lower than that of the wild type, indicating that the homologous recombination of the mtopVIB mutant was also abnormal. The phenotype of the mutant mtopVIB-2 and the DSB repair defect mutants Atcom1-1, Atrad50-1, Atrad51 and Atmre11-1 were constructed, and the phenotype of these double mutants and the mtopVIB-2 process were the same. The phenotypic similarity of the variant is similar to that of the monovalent in the meiosis process, indicating that mtopVIB-2 does affect the formation of DSB, indicating that MTOPVIB participates in the formation of meiotic DSB,.Real-time PCR analysis indicates that MTOPVIB is formed in group form, and that the higher expression of.MTOPVIB in the inflorescence belongs to a class of conserved protein, and its structure and To The B subunit of the Po VI complex is similar, and its N terminal contains a Bergerat domain that can bind and hydrolyze ATP. The C end contains a Transducer domain that can induce conformation changes,.Y2H and BiFC experimental results show that MTOPVIB protein can be interacted with AtSPO11-1 and protein respectively. At the same time interacting with them, it is suggested that a Topo VI similar complex.Y2H and BiFC experiment may be formed among the three, and that AtPRD1 can interact with MTOPVIB and AtSP011-2 respectively, indicating that AtPRD1 can interact with the members of the Topo VI similar complex. In addition, Y3H and BiFC experiments show that AtPRD1 protein can mediate interaction with AtPRD3 and AtDFO proteins. This indicates that AtPRD1 is likely to be an important protein in the formation of DSB. To sum up, in Arabidopsis, MTOPVIB protein is interacted with AtPRD1 protein and participates in Arabidopsis together with AtSPO11-1, AtSP011-2, AtPRD3, and such proteins. DSB results in the process of gametophyte meiosis. These results provide new experimental evidence for understanding the mechanism of meiosis DSB formation in Arabidopsis thaliana.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

【参考文献】